Collaborative Research: Effects of exopolymeric substances (EPS) on engineered nanoparticle (EN) into marine phytoplankton cells

0933137/0932404 Santschi/ ChinEngineered Nanoparticles（ENs）的尺寸范围为1至100 nm，具有与相同化学成分的散装材料不同的特性，用于制造许多消费品，包括印刷涂料、洗涤剂、杀菌剂、涂料、化妆品、防晒霜、轮胎、计算机电子产品和药物输送。 尽管这些材料的使用日益广泛，但其对人类健康和海洋生态系统健康的影响在很大程度上是未知的。 EN包括碳纳米管、含金属颗粒、零价金属产物、树枝状聚合物和量子点（QD）。 拟议的研究将利用量子点，因为它们不仅在生物成像，疾病诊断和治疗中有许多应用，而且它们的宽激发光谱，可调谐发射波长和非常稳定的荧光也使它们适合于机理研究。 虽然EN的主要问题是它们的潜在毒性（例如，产生活性氧的潜力），拟议的研究将主要集中在海洋浮游植物细胞的运输步骤和机制，这可能受到静电和疏水相互作用的影响。 例如，EN的相对亲水性是控制其到达沿海沃茨的能力以及其生物利用度和生物吸收的关键因素之一。由于陆地和水生天然有机物质如富里酸和外聚物（EPS）的乳化和表面活性剂性质，可以预期到达表面沃茨的EN更亲水。海洋中浮游植物和细菌分泌的EPS是富含多糖的阴离子胶体聚合物，其在海洋凝胶、海洋雪和生物膜的形成中以及在胶体和微量元素清除中以及在提供针对病毒感染的保护中是重要的。相同的EPS特性也会影响EN在生物体中的吸收和积累。基于对动物细胞的有限研究，EN可以通过内吞或内吞进入细胞，（待摄入的物质逐渐被质膜包围，质膜最终分离形成内吞囊泡）或通过其他非载体相关过程如表面粘附和被动扩散。EPS对水生系统中EN稳定性的影响以及EN穿过海洋浮游植物膜的传输机制将被研究。在拟议的研究中将检验以下假设：H1：QD在水环境中的稳定性可受到EPS的显著影响，EPS可改变QD进入细胞的附着和/或进入途径，其中EPS的相对疏水性起主要作用。H2：量子点不仅可以吸附在细胞表面，但也可以内化和集中在某些藻类细胞器;这反过来又将决定其最终的影响（消除或改善潜在的损害）浮游植物。H3：量子点可能通过内吞作用进入无明显细胞壁的浮游植物体内，而非囊泡相关过程可能是量子点进入有细胞壁的浮游植物体内的主要机制。或者量子点可能不一定进入细胞，但刺激非特异性膜受体。H4：细胞内Ca 2+信号在多种细胞反应的刺激偶联激活中起着普遍的关键作用。量子点与浮游植物细胞的潜在相互作用可以触发细胞内Ca 2+升高，导致胞吐和/或胞吞作用。 为了检验这四个假设，五种不同的浮游植物将首先筛选其产生EPS和细胞内吸收ENs的能力。经过初步筛选，五个物种中的两个将被选择为进一步的机制研究，他们与量子点，这将是EN的选择。此外，本研究亦将探讨内分泌物的亚细胞分布，以及内分泌物和胞外聚合物通过Ca ~（2+）介导的分泌过程从浮游植物细胞中排出的情况，除了有助于我们了解内分泌物在水生系统中的行为和毒性、浮游植物的毒性反应，以及胞外聚合物的相对疏水性/亲水性在改变内分泌物的稳定性和归宿方面的重要性外，这项研究的结果也将有助于ENs法规政策制定的知识基础，并将加强TAMUG和加州大学默塞德的博士后研究员、研究生和本科生的培训计划。它还将有助于提高科学界、学术界和地方社区对与这一发展中技术有关的环境风险的认识。

0933137/0932404Santschi/ ChinEngineered Nanoparticles (ENs), which range in size from 1 to 100 nm and have properties that are different from those of bulk materials of the same chemical composition, are used in the fabrication of numerous consumer goods, including printing paints, detergents, bactericides, coatings, cosmetics, sunscreens, tires, computer electronics, and drug delivery. In spite of their growing and widespread use, the impacts of these materials on human health and the health of ocean ecosystems are largely unknown. ENs include carbon nanotubes, metal containing particles, zero-valent metal products, dendrimers, and quantum dots (QDs). The proposed research will utilize QDs, as they not only have many applications in biological imaging, disease diagnostics, and therapeutics, but their broad excitation spectrum, tunable emission wavelength and quite stable fluorescence also make them suitable for mechanistic studies. While the major concern with ENs is in terms of their potential toxicity (e.g., the potential for producing reactive oxygen species), the proposed research will mainly focus on the transport steps and mechanisms into marine phytoplankton cells, which can be affected by both electrostatic and hydrophobic interactions. For example, the relative hydrophilicity of ENs is one of the key factors controlling their ability to reach coastal waters, as well as their bioavailability and biological uptake. Because of the emulsification and surfactant qualities of terrestrial and aquatic natural organic substances such as fulvic acids and exopolymeric substances (EPS), it can be expected that ENs that reach surface waters are more hydrophilic. EPS, secreted from phytoplankton and bacteria in the ocean are polysaccharide-rich anionic colloidal polymers that are important in the formation of marine gels, marine snow and biofilms, as well as in colloid and trace element scavenging and in providing protection against virus infection. The same EPS characteristics will also affect EN uptake and accumulation in organisms. Based on the limited research on animal cells, ENs can enter cells either by endocytosis (material to be ingested is progressively enclosed by the plasma membrane, which eventually detaches to form an endocytic vesicle) or by other non-vehicle related processes such as surface adhesion and passive diffusion.In this research, the effects of EPS on EN stability in aquatic systems and the mechanisms of EN transport across the marine phytoplankton membranes will be examined. The following hypothesis will be tested in the proposed research: H1: QD stability in the aquatic environment can be significantly influenced by EPS, which can alter the attachment and/or entry pathways of QDs into cells, with the relative the hydrophobicity of EPS playing a major role. H2: QDs may not only be adsorbed onto the cell surface but may also be internalized and concentrated in certain algal organelles; this in turn will determine their final effects (either elimination or amelioration of potential damages) on phytoplankton. H3: QDs may enter phytoplankton without an obvious cell wall through endocytosis while non-vesicle related processes may be the main mechanisms of entry in phytoplankton with cell walls. Or QDs may not necessarily enter cells, but stimulate nonspecifically membrane receptors. H4: Intracellular Ca2+ signaling plays a universal critical role in stimulus coupling of activation in a broad variety of cell responses. The potential interactions of QDs with phytoplankton cells could trigger intracellular Ca2+ elevation leading to exocytosis and/or endocytosis. In order to test these four hypotheses, five different phytoplankton species will be screened first for their ability to produce EPS and to uptake ENs intracellularly. After an initial screening, two of the five species will be chosen for further mechanistic research on their interactions with QDs, which will be the EN of choice. EN subcellular distribution as well as the excretion of ENs and EPS out of the phytoplankton cells via a Ca2+-mediated secretion process will also be examined.Besides contributing to our knowledge of ENs' behavior and toxicity in aquatic systems, toxicity responses of phytoplankton, and importance of relative hydrophobicity/hydrophilicity of EPS in changing stability and fate of ENs, the outcome of this research will also contribute to the knowledge base for policy-making of ENs regulations, and will enhance training programs at TAMUG and UC Merced for postdoctoral fellows, graduate and undergraduate students. It will also contribute to increased awareness in the scientific, academic and local communities of the environmental risks associated with this developing technology.